When light is absorbed within the outer segment of a vertebrate photoreceptor, the conformation of the photopigment rhodopsin is altered to produce an activated photoproduct called metarhodopsin II or Rh(*). Rh(*) initiates a transduction cascade similar to that for metabotropic synaptic receptors and many hormones; the Rh(*) activates a heterotrimeric G protein, which in turn stimulates an effector enzyme, a cyclic nucleotide phosphodiesterase. The phosphodiesterase then hydrolyzes cGMP, and the decrease in the concentration of free cGMP reduces the probability of opening of channels in the outer segment plasma membrane, producing the electrical response of the cell. Photoreceptor transduction can be modulated by changes in the mean light level. This process, called light adaptation (or background adaptation), maintains the working range of the transduction cascade within a physiologically useful region of light intensities. There is increasing evidence that the second messenger responsible for the modulation of the transduction cascade during background adaptation is primarily, if not exclusively, Ca(2+), whose intracellular free concentration is decreased by illumination. The change in free Ca(2+) is believed to have a variety of effects on the transduction mechanism, including modulation of the rate of the guanylyl cyclase and rhodopsin kinase, alteration of the gain of the transduction cascade, and regulation of the affinity of the outer segment channels for cGMP. The sensitivity of the photoreceptor is also reduced by previous exposure to light bright enough to bleach a substantial fraction of the photopigment in the outer segment. This form of desensitization, called bleaching adaptation (the recovery from which is known as dark adaptation), seems largely to be due to an activation of the transduction cascade by some form of bleached pigment. The bleached pigment appears to activate the G protein transducin directly, although with a gain less than Rh(*). The resulting decrease in intracellular Ca(2+) then modulates the transduction cascade, by a mechanism very similar to the one responsible for altering sensitivity during background adaptation.

Using suction electrodes, photocurrent responses to 100-ms saturating flashes were recorded from isolated retinal rods of the larval-stage tiger salamander (Ambystoma tigrinum). The delay period (7" c ) that preceded recovery of the dark current by a criterion amount (3 pA) was analyzed in relation to the flash intensity (If), and to the corresponding fractional bleach (R* 0 /R lol ) of the visual pigment; Rl/R lol was compared with R*/R lol , the fractional bleach at which the peak level of activated transducin approaches saturation. Over an approximately 8 In unit range of If that included the predicted value of R*/R lol , T c increased linearly with In If. Within the linear range, the slope of the function yielded an apparent exponential time constant (T C ) of 1.7 ± 0.2 s (mean ± S.D.). Background light reduced the value of T c measured at a given flash intensity but preserved a range over which T c increased linearly with In If-, the linear-range slope was similar to that measured in the absence of background light. The intensity dependence of T c resembles that of a delay (T d ) seen in light-scattering experiments on bovine retinas, which describes the period of essentially complete activation of transducin following a bright flash; the slope of the function relating T d and In flash intensity is thought to reflect the lifetime of photoactivated visual pigment (/?*) (Pepperberg et al., 1988; Kahlert et al., 1990). The present data suggest that the electrophysiological delay has a similar basis in the deactivation kinetics of / ? ' , and that T C represents T R >, the lifetime of R* in the phototransduction process. The results furthermore suggest a preservation of the "dark-adapted" value of T R * within the investigated range of background intensity.

1. We have used suction electrode recording together with rapid steps into Li' solution and 0 5 mM IBMX solution to estimate the rates of the guanylyl phosphodiesterase (PDE) and guanylyl cyclase in isolated rods of the salamander, Ambystoma tigrinum. 2. We show that both the PDE and cyclase velocities are accelerated by steady background light. The steady velocities of both enzymes appear to be saturating functions of background intensity. 3. Bleaching also accelerates both the PDE and cyclase. This effect is maintained long after the bleaching stimulus is removed (up to 2 h) and is reversed only if the photopigment is regenerated with exogenous chromophore. 4. The estimated steady-state PDE and cyclase velocities appear to be linear functions of the amount of pigment bleached, as if each bleached pigment molecule activated the transduction cascade with the same probability and gain. 5. The effectiveness of bleached pigment in activating transduction is only 10-6 to times that of activated rhodopsin (Rh*), but this is sufficient after large bleaches to produce an 'equivalent background' excitation of the rod, which is probably responsible, at least in part, for bleaching desensitization.When the eye is exposed to light bright enough to bleach a significant fraction of the visual pigment, the sensitivity of the visual system is greatly depressed and recovers slowly. This phenomenon, known as dark adaptation or bleaching adaptation, is still poorly understood. Since the recovery of sensitivity has a time course which is approximately the same as the regeneration of visual pigment (Rushton, 1965), it is reasonable to suppose that the loss of pigment is somehow responsible for the loss of sensitivity. However, it has long been realized that the decrease in sensitivity is much greater than would be expected from the decrease in the probablility of light absorption by the photopigment (Campbell & Rushton, 1955). The visual system appears to produce a bleaching signal that elevates threshold much more than simple loss of pigment would predict.To investigate the nature of this bleaching signal, we have recorded from isolated rods of the salamander, Ambystoma tigrinum. Earlier experiments have shown that the bleaching of pigment in these photoreceptors produces a loss of sensitivity which, like that in the visual system as a whole, is considerably greater than expected from the decrease in pigment concentration (Cornwall, Fein & MacNichol, 1990). Furthermore, the light responses of bleached rods at steady state have many of the characteristics of those of rods in the presence of background light: sensitivity is decreased, circulating current is reduced, and the decay time of the response to brief flashes is accelerated. These observations suggest that bleaching may desensitize the rods by producing an 'equivalent background' excitation, as first suggested from psychophysical experiments by Stiles & Crawford (1932).Furthermore, the similarity of responses in the presence of background light and after bleaches suggests t...

After visual-pigment bleaching, single isolated rod photoreceptors of Ambystoma tigrinum recover their sensitivity to light when supplied with 11-cis-retinal from liposomes or with li-cis-retinal bound to interphotoreceptor retinoid-binding protein. Bleached rods do not recover sensitivity, or do so only very slowly, after exposure to 11-cis-retinol. The latter retinoid is "toxic" in that rods actually lose sensitivity in its presence. In contrast, bleached isolated cone cells recover sensitivity when either retinoid is supplied. It is suggested that the major pathway for rhodopsin regeneration during dark adaptation in the intact eye is transport of ll-cis-retinal from the pigment epithelium to the retina. The results also suggest that there may be separate pathways for visual-pigment regeneration in rods and cones during dark adaptation.Regeneration of visual pigment during dark adaptation or during maintained illumination requires retinoid isomerization from trans to cis form and conversion from alcohol to aldehyde before retinoid can be bound to opsin to reconstitute active pigment (1). The pigment epithelium (PE) has long been known to be involved in regeneration (2), implying a "visual cycle" that involves shuttling of retinoid from retina to PE and back again during cycles of light and dark. During light adaptation, there is indeed a progressive loss of retinoid from the retina, and an increase in the retinoid content of the PE. During darkness, the retinoid flow is reversed (3). Fulton and Rando (4) have presented strong evidence for localization of the retinoid-isomerizing system in PE rather than retina, but it remains uncertain which cells are involved in effecting the alcohol-to-aldehyde change that must take place during the visual cycle.We now report a difference in the use of retinol and retinal by rod and cone cells, and a "toxic" effect of retinol on rod cell function. We suggest that 11-cis-retinal is the major form of retinoid transported from PE to retina. Alternatively, ifthe alcohol form is transported it must be converted to the aldehyde in a cell type other than the rod photoreceptor (which may be cones, or possibly Muller cells).Pepperberg et al. (5) showed that the ordinarily permanent desensitization due to bleaching in isolated retina could be reversed by treatment with 11-cis-retinal. In their work, retinal was applied in ethanolic solution. Subsequently, it was shown (6) that liposomes could also be used to deliver retinoid. Our studies on isolated photoreceptors have employed both liposomes and interphotoreceptor retinoidbinding protein (IRBP). IRBP is a protein found at high concentration in the interphotoreceptor matrix, where its unique location and retinoid-binding properties (7,8) make it likely that it is involved in retinoid movement between retina and PE. In support of this, we demonstrate here the transfer of retinoid between IRBP and photoreceptor cells. MATERIALS AND METHODSIsolated photoreceptor cells from dark-adapted, larval tiger salamanders (Ambystoma tigrin...

A spot confocal microscope based on an argon ion laser was used to make measurements of cytoplasmic calcium concentration (Ca2+ i) from the outer segment of an isolated rod loaded with the fluorescent calcium indicator fluo-3 during simultaneous suction pipette recording of the photoresponse. The decline in fluo-3 fluorescence from a rod exposed to saturating illumination was best fitted by two exponentials of approximately equal amplitude with time constants of 260 and 2,200 ms. Calibration of fluo-3 fluorescence in situ yielded Ca2+ i estimates of 670 ± 250 nM in a dark-adapted rod and 30 ± 10 nM during response saturation after exposure to bright light (mean ± SD). The resting level of Ca2+ i was significantly reduced after bleaching by the laser spot, peak fluo-3 fluorescence falling to 56 ± 5% (SEM, n = 9) of its value in the dark-adapted rod. Regeneration of the photopigment with exogenous 11-cis-retinal restored peak fluo-3 fluorescence to a value not significantly different from that originally measured in darkness, indicating restoration of the dark-adapted level of Ca2+ i. These results are consistent with the notion that sustained activation of the transduction cascade by bleached pigment produces a sustained decrease in rod outer segment Ca2+ i, which may be responsible for the bleach-induced adaptation of the kinetics and sensitivity of the photoresponse.

Summary Daytime vision is mediated by retinal cones which, unlike rods, remain functional even in bright light and dark-adapt rapidly. These cone properties are enabled by rapid regeneration of their pigment. This in turn requires rapid chromophore recycling which may not be achieved by the canonical retinal pigment epithelium visual cycle. Recent biochemical studies have suggested the presence of a second, cone-specific visual cycle, although its physiological function remains to be established. Here we report that the Müller cells within the salamander neural retina promote cone-specific pigment regeneration and dark adaptation that are independent of the pigment epithelium. Without this pathway, dark adaptation of cones is slow and incomplete. Interestingly, the rates of cone pigment regeneration by the retina and pigment epithelium visual cycles are essentially identical suggesting a possible common rate-limiting step. Finally, we also observed cone dark adaptation in the isolated mouse retina.

Simultaneous measurements of photocurrent and outer segment Ca2+ were made from isolated salamander cone photoreceptors. While recording the photocurrent from the inner segment, which was drawn into a suction pipette, a laser spot confocal technique was employed to evoke fluorescence from the outer segment of a cone loaded with the Ca2+ indicator fluo-3. When a dark-adapted cone was exposed to the intense illumination of the laser, the circulating current was completely suppressed and fluo-3 fluorescence rapidly declined. In the more numerous red-sensitive cones this light-induced decay in fluo-3 fluorescence was best fitted as the sum of two decaying exponentials with time constants of 43 ± 2.4 and 640 ± 55 ms (mean ± SEM, n = 25) and unequal amplitudes: the faster component was 1.7-fold larger than the slower. In blue-sensitive cones, the decay in fluorescence was slower, with time constants of 140 ± 30 and 1,400 ± 300 ms, and nearly equal amplitudes. Calibration of fluo-3 fluorescence in situ from red-sensitive cones allowed the calculation of the free-Ca2+ concentration, yielding values of 410 ± 37 nM in the dark-adapted outer segment and 5.5 ± 2.4 nM after saturating illumination (mean ± SEM, n = 8). Photopigment bleaching by the laser resulted in a considerable reduction in light sensitivity and a maintained decrease in outer segment Ca2+ concentration. When the photopigment was regenerated by applying exogenous 11-cis-retinal, both the light sensitivity and fluo-3 fluorescence recovered rapidly to near dark-adapted levels. Regeneration of the photopigment allowed repeated measurements of fluo-3 fluorescence to be made from a single red-sensitive cone during adaptation to steady light over a range of intensities. These measurements demonstrated that the outer segment Ca2+ concentration declines in a graded manner during adaptation to background light, varying linearly with the magnitude of the circulating current.

Retinal rod and cone pigments consist of an apoprotein, opsin, covalently linked to a chromophore, 11-cis retinal. Here we demonstrate that the formation of the covalent bond between opsin and 11-cis retinal is reversible in darkness in amphibian red cones, but essentially irreversible in red rods. This dissociation, apparently a general property of cone pigments, results in a surprisingly large amount of free opsin--about 10% of total opsin--in dark-adapted red cones. We attribute this significant level of free opsin to the low concentration of intracellular free 11-cis retinal, estimated to be only a tiny fraction (approximately 0.1 %) of the pigment content in red cones. With its constitutive transducin-stimulating activity, the free cone opsin produces an approximately 2-fold desensitization in red cones, equivalent to that produced by a steady light causing 500 photoisomerizations s-1. Cone pigment dissociation therefore contributes to the sensitivity difference between rods and cones.